Functional Role of Pseudouridine on Zika Virus Gene Expression

假尿苷对寨卡病毒基因表达的功能作用

• 批准号: 10732617
• 负责人: Cara Theresia Pager
• 金额: $ 22.8万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT ALBANY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-09 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10732617
• 关键词: Affect; Affinity; Americas; Antisense Oligonucleotides; Antiviral Agents; Appearance; Biological Assay; Biotinylation; Cell Line; Cell Nucleus; Cells; Chemicals; Culicidae; Cytoplasm; Defect; Detection; Development; Disease Outbreaks; Enzymes; Flavivirus; Gene Expression; Genome; Goals; HIV-1; Health; Hepatitis C virus; Human; Immune response; Infection; Knowledge; Label; Length; Life Cycle Stages; Luciferases; Mass Spectrum Analysis; Methodology; Microcephaly; Modification; Molecular; Molecular Biology; Mutagenesis; Neurologic; Nuclear Export; Nucleotides; Plaque Assay; Polyribosomes; Positioning Attribute; Proteins; Pseudouridine; RNA; RNA Folding; RNA Interference; RNA Stability; RNA Viruses; RNA-Protein Interaction; Regulation; Replicon; Reporting; Research; Reverse Transcription; Role; Site; Site-Directed Mutagenesis; Stress; Structure; System; Techniques; Technology; Testing; Therapeutic; Time; Transcript; Translations; Untranslated Regions; Viral; Viral Genes; Viral Physiology; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Western Blotting; Woman; ZIKA; ZIKV infection; Zika Virus; combinatorial; epitranscriptomics; hepatoma cell; improved; infancy; insight; mosquito-borne; new therapeutic target; next generation sequencing; novel; overexpression; therapeutic target; transcriptome sequencing; viral RNA; viral transmission

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus, that during the 2015-2016 outbreak in the Americas was associated with severe and devastating developmental abnormalities, including microcephaly, in babies born from infected women. The single-stranded positive-sense RNA genome of ZIKV directs translation of viral proteins, functions as a template for replication of the genome and is packaged into new virus particles. These functions are in part regulated by specific RNA structures within the untranslated regions of the RNA, and via interactions with viral and cellular proteins. More recently RNA modifications have been shown to significantly affect the virus infectious cycle. Although more than 170 different RNA modifications are known to decorate natural RNAs, only N6-methyladenosine has been reported to affect ZIKV gene expression. The role of other RNA modifications on ZIKV RNA is presently unknown. Using biotinylated antisense oligonucleotides to affinity isolate virus RNA from cells and virions, and mass spectrometry to identify RNA modifications, we discovered that more than 30 different RNA modifications are present on ZIKV RNA. In this application we focus on pseudouridine (Y), an abundant but understudied RNA modification. The incorporation of Y on cellular RNAs affects the folding, stability, and translation of RNA, and RNA-RNA and RNA-protein interactions. We propose that Y on the ZIKV RNA genome promotes virus translation and replication. In this application we propose using a next generation sequencing approach combined with chemical modification of pseudouridine to identify specific Y sites on ZIKV genome (Aim 1). Next, we will undertake RNAi depletion and overexpression of cellular pseudouridine synthase proteins to identify the host enzymes responsible for installing pseudouridine on ZIKV RNA (Aim 2). Last, we will use mutagenesis and modulation of the pseudouridine synthase enzymes to elucidate if pseudouridine regulates ZIKV translation and/or replication (Aim 3). Moreover, these studies will be undertaken in the context of virus infection in mammalian and mosquito cells, two hosts biologically relevant to the virus life cycle, which could inform the role of RNA modifications in virus transmission and host adaptation. We expect that this research will advance our knowledge of RNA modifications which have been shown to be novel and important regulators of viral gene expression and improve our understanding of the molecular biology of ZIKV. This research will therefore provide critical information on a virus with significant impacts on human health and that currently lacks therapeutic options.

寨卡病毒（ZIKV）是一种重新出现的蚊媒黄病毒，在2015-2016年美洲爆发期间，与受感染妇女所生婴儿的严重和毁灭性发育异常有关，包括小头畸形。ZIKV的单链正义RNA基因组指导病毒蛋白的翻译，充当基因组复制的模板，并包装成新的病毒颗粒。这些功能部分地由RNA的非翻译区内的特定RNA结构以及通过与病毒和细胞蛋白的相互作用来调节。最近，RNA修饰已显示出显著影响病毒感染周期。尽管已知有超过170种不同的RNA修饰天然RNA，但据报道只有N6-甲基腺苷影响ZIKV基因表达。其他RNA修饰对ZIKV RNA的作用目前尚不清楚。使用生物素化的反义寡核苷酸从细胞和病毒体中亲和分离病毒RNA，并使用质谱法鉴定RNA修饰，我们发现ZIKV RNA上存在超过30种不同的RNA修饰。在本申请中，我们专注于假尿苷（Y），一种丰富但研究不足的RNA修饰。Y在细胞RNA上的掺入影响RNA的折叠、稳定性和翻译，以及RNA-RNA和RNA-蛋白质相互作用。我们认为ZIKV RNA基因组上的Y促进病毒翻译和复制。在本申请中，我们提出使用与假尿苷的化学修饰组合的下一代测序方法来鉴定ZIKV基因组上的特异性Y位点（目的1）。接下来，我们将进行细胞假尿苷合酶蛋白的RNAi耗尽和过表达，以鉴定负责在ZIKV RNA上安装假尿苷的宿主酶（Aim 2）。最后，我们将使用假尿苷合酶的诱变和调节来阐明假尿苷是否调节ZIKV翻译和/或复制（目的3）。此外，这些研究将在哺乳动物和蚊子细胞中的病毒感染的背景下进行，这两种宿主与病毒生命周期具有生物学相关性，这可以为RNA修饰在病毒传播和宿主适应中的作用提供信息。我们希望这项研究将推进我们对RNA修饰的了解，这些修饰已被证明是病毒基因表达的新的和重要的调节因子，并提高我们对ZIKV分子生物学的理解。因此，这项研究将提供有关对人类健康有重大影响且目前缺乏治疗方案的病毒的关键信息。